A method for detecting beneficial activity based on multiple strains

Abstract

The present invention provides a method for detecting the probiotic activity based on multiple strains, including selecting strains to be detected for probiotic activity, the strains including beneficial bacteria and pathogenic bacteria, culturing the selected strains with the prebiotic to be evaluated as the only carbon source of the culture medium, using glucose carbon source culture medium as a positive control group, and sugar-free culture medium as a blank group, obtaining the growth values ​​of all strains and the utilization values ​​of prebiotics, and calculating the probiotic activity. The present invention compares and cultures multiple strains to expand the strain coverage, is more objective and accurate than a single strain, and reduces the contingency and specificity in the detection process. In addition to performing culture detection of multiple groups of probiotics and potential pathogens, the present invention also provides a calculation of the utilization of prebiotics by the strains. At the same time, the present invention uses absorbance at 600nm instead of colony counts for calculation, and calculates the growth value immediately after the strain culture is completed, thereby improving the detection calculation efficiency and facilitating its promotion and application.

Description

Technical Field

[0001] The present invention relates to the technical field of prebiotics, and in particular to a prebiotic activity detection method based on multiple strains. Background Art

[0002] There are many types of prebiotics, such as galacto-oligosaccharides, fructo-oligosaccharides, inulin, xylo-oligosaccharides, 2'-fucosyllactose, etc. Currently, a representative probiotic and a representative intestinal bacteria are used, with glucose, which promotes both beneficial bacteria and pathogenic bacteria, as a positive control. The test calculation is performed according to the following formula: and Represents the viable colony-forming units (CFU) / mL of beneficial bacteria cultured on prebiotics at 24h and 0h, and Represents the viable colony-forming units (CFU) / mL of beneficial bacteria cultured on glucose at 24h and 0h, and Represents the viable colony-forming units (CFU) / mL of intestinal bacteria cultured on prebiotics at 24h and 0h, and The score represents the number of viable colony-forming units (CFU) / mL of intestinal bacteria cultured on glucose at 24 hours and 0 hours of incubation. The scoring design requires that prebiotics promote the growth of beneficial bacteria while not supporting the growth of pathogenic bacteria, reflecting the "selectivity" in the definition of prebiotics.

[0003]

[0004] However, scoring based solely on a single strain is too subjective. There are many types of beneficial intestinal bacteria (Bifidobacterium, Lactobacillus, Bacillus, etc.) and pathogenic bacteria (Enterobacter sakazakii, Campylobacter jejuni, Klebsiella, etc.), so the results of prebiotic testing calculations depend entirely on the selected strain. Moreover, the formula reflects the number of probiotics from the surface and cannot intuitively reflect whether the prebiotics are being utilized by the probiotics. In addition, the experimental cycle of viable colony-forming units is long, the experimental accuracy requirements are high, and errors are prone to occur. Therefore, reflecting the activity of prebiotics by testing multiple strains of probiotics and multiple strains of pathogens is a more effective and accurate method, which can comprehensively and horizontally compare the probiotic activity of each prebiotic. Summary of the Invention

[0005] The purpose of the present invention is to provide a multi-strain based method for detecting the activity of prebiotics, which can reflect the activity of prebiotics by detecting and calculating multiple strains of probiotics and multiple strains of pathogenic bacteria, which is a more effective and accurate way to comprehensively and horizontally compare the probiotic activities of various prebiotics.

[0006] To achieve the above objectives, the present invention proposes the following technical solution: a method for detecting the beneficial activity of multiple strains, comprising the following steps:

[0007] Step 1: selecting strains to be tested for beneficial activity, the strains including beneficial bacteria and pathogenic bacteria, and the number of the strains is greater than 3;

[0008] Step 2: The selected strains were cultured using the prebiotic to be evaluated as the sole carbon source in the culture medium, with glucose carbon source culture medium as the positive control group and sugar-free culture medium as the blank group;

[0009] Step 3: Obtain the growth values ​​of all strains in step 2, draw a growth curve based on the OD600 absorbance value at each time point during the culture process, calculate the area under the growth curve, and then calculate the growth value of each strain;

[0010] Step 4: Obtain the prebiotic utilization value of the strain, perform thin layer chromatography analysis on the supernatant of the bacterial liquid before and after cultivation of each strain, calculate the prebiotic consumption in the thin layer chromatography spectrum, and further calculate the prebiotic utilization value of each strain;

[0011] Step 5: Calculate the prebiotic activity using the growth value in step 3 and the prebiotic utilization value in step 4.

[0012] Preferably, the beneficial bacteria include one or more of Bifidobacterium, Lactobacillus, Lactococcus, Streptococcus, Clostridium, and Enterococcus, and the pathogenic bacteria include one or more of Escherichia coli, Salmonella, Staphylococcus, Klebsiella, Vibrio, or Bacillus.

[0013] Preferably, the calculation method of the strain growth value in step 3 is as follows: the total number of strains is n, the strain number is i, 1≤i≤n, the prebiotic is named X, the blank group is named M, and the OD of the strain i at 0h, 12h, 24h, 36h, 48h until the fermentation end point th is calculated. 600 The growth curve was drawn with the OD at 0 h. 600 The value is the baseline, and the area under the integrated 0-th growth curve is the value The growth value G is the area of ​​the prebiotic group (X) minus the area of ​​the blank group (M). i ;

[0014]

[0015] Preferably, the calculation method of the prebiotic utilization value in step 4 is as follows:

[0016] The total number of strains is n, the strain number is i, the prebiotic is named X, and thin layer chromatography is performed at fermentation 0h and fermentation end point th. The optical density value of th in the thin layer chromatography of the prebiotic is calculated using Image J software. and optical density value at 0h Subtract and divide by the optical density value at 0h Prebiotic utilization value U i ;

[0017]

[0018] Preferably, the calculation method of the beneficial activity in step 5 is as follows: the total number of beneficial bacteria is n, each strain is numbered i, the total number of potential pathogens is N, each strain is numbered k, and the mean of the beneficial bacteria growth value is Divide by the mean of the potential pathogen growth value The average utilization value of beneficial bacteria and prebiotics Divided by the mean of the prebiotic utilization value of potential pathogens The prebiotic activity value P can be obtained;

[0019]

[0020] Beneficial effects: The technical solution of the present application has the following technical effects: the present invention expands the strain coverage by culture detection and comparison of multiple strains, which is more objective and accurate than a single strain, and reduces the randomness and specificity in the detection process. In addition to culture detection of multiple groups of probiotics and potential pathogenic bacteria, the present invention also provides a calculation of the utilization of prebiotics by strains. At the same time, the present invention uses the absorbance value at 600nm instead of the colony count (CFU) for calculation, and can calculate the growth value immediately after the strain culture is completed, thereby improving the detection calculation efficiency and facilitating its promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] in:

[0022] Figure 1 Flowchart of the present invention.

[0023] Figure 2 This is the growth curve of Bifidobacterium under different carbon source conditions in Example 2.

[0024] Figure 3 This is the growth curve of Lactobacillus under different carbon source conditions in Example 2.

[0025] Figure 4 This is the growth curve of Clostridium butyricum under different carbon source conditions in Example 2.

[0026] Figure 5 This is the growth curve of Enterococcus faecalis under different carbon source conditions in Example 2.

[0027] Figure 6 This is the growth curve of Escherichia coli under different carbon source conditions in Example 2.

[0028] Figure 7 This is the growth curve of Salmonella under different carbon source conditions in Example 2.

[0029] Figure 8 This is the growth curve of Klebsiella under different carbon source conditions in Example 2.

[0030] Figure 9 This is the growth curve of Clostridium perfringens under different carbon source conditions in Example 2.

[0031] Figure 10 This is the TLC diagram of different prebiotics fermented by various strains in Example 3. Figure 10 Figure A in the figure is glucose. Figure 10 Figure B in the figure is 2'-fucosyllactose, Figure 10 Figure C in the figure is fucose, Figure 10 Figure D in the figure is fucoidan oligosaccharide. Figure 10 Figure E in the figure is galacto-oligosaccharide.

[0032] Figure 11 It is an AUC (area under the growth curve) graph.

[0033] Figure 12 This is the IntDen (optical density value) graph. DETAILED DESCRIPTION

[0034] In order to better understand the technical content of the present invention, specific embodiments are given and described as follows in conjunction with the accompanying drawings. Various aspects of the present invention are described in this disclosure with reference to the accompanying drawings, in which many illustrative embodiments are shown. The embodiments of the present disclosure are not necessarily defined to include all aspects of the present invention. It should be understood that the various concepts and embodiments introduced above, as well as those described in more detail below, can be implemented in any of many ways, because the concepts and embodiments disclosed in the present invention are not limited to any implementation method. In addition, some aspects disclosed in the present invention can be used alone or in any appropriate combination with other aspects disclosed in the present invention.

[0035] Example 1 Pure culture of strains

[0036] Four prebiotics were selected: galacto-oligosaccharides, fucoidan oligosaccharides, 2'-fucosyllactose and fucose. Glucose was used as the positive control group, and sugar-free culture medium was used as the blank group. Four probiotic strains were selected: one Bifidobacterium, one Lactobacillus, one Clostridium butyricum, and one Enterococcus faecalis. Four potential pathogens were selected: one Escherichia coli, one Salmonella, one Klebsiella, and one Clostridium perfringens. The OD value of the bacterial solution was measured every 12 hours starting from the initial 0 hour using a microplate reader. 600 The supernatant was used for thin layer chromatography.

[0037] Example 2 Based on OD 600 The area under the growth curve (AUC) was calculated.

[0038] Growth value determination: OD at 0h, 12h, 24h, 36h, and 48h600 Draw a growth curve, such as Figure 2-11 The area under the curve (AUC; OD 600 ×min). The AUC value of prebiotic X minus the AUC value of blank group M is the growth value of the strain, and the formula is as follows:

[0039]

[0040] The growth curves and growth values ​​of each strain indicate that the same prebiotic induces different growth responses in different strains. Glucose has a strong growth-promoting effect on both beneficial and potentially pathogenic bacteria, while galacto-oligosaccharides not only significantly promote the growth of beneficial bacteria but also partially promote the growth of potentially pathogenic bacteria. Fucoido-oligosaccharides promote the growth of three strains of beneficial bacteria but rarely support the growth of potentially pathogenic bacteria. 2'-fucosyllactose is only utilized by one strain of beneficial bacteria but strictly does not support the growth of potentially pathogenic bacteria. Fucose fails to promote the proliferation of most strains and even partially inhibits the growth of Enterococcus faecalis and Escherichia coli.

[0041] Table 1 Growth values ​​of each strain

[0042]

[0043] Example 3 was calculated based on the optical density value (Den) of thin layer chromatography (TLC).

[0044] Determination of prebiotic utilization value: TLC analysis was performed on the supernatant of 0h and 48h fermentation. The TLC spectrum was as follows: Figure 10 The TLC spectra were converted into black and white optical density images using Image J ( Figure 12 ) and calculate the optical density values ​​of the prebiotic at 0 h and 48 h. The optical density difference is the optical density value of prebiotic X at 48 h minus the optical density value at 0 h. This difference is then divided by the optical density value at 0 h to obtain the prebiotic utilization value. The formula is as follows:

[0045]

[0046] The TLC profiles and prebiotic utilization values ​​of each strain indicate that different strains utilize the same prebiotic to varying degrees. Glucose is fully utilized by all beneficial and potential pathogenic bacteria, with utilization values ​​of 100%. Although galacto-oligosaccharides can be utilized by potential pathogens, the beneficial bacteria Lactobacillus exhibits the highest utilization. Fuco-oligosaccharide is utilized by four beneficial bacteria, while two potential pathogens can utilize it, albeit at a lower rate. 2'-fucosyllactose is utilized by only two beneficial strains and partially by only one potential pathogen. Fucose is not utilized by most strains, but is fully utilized by the potential pathogens Salmonella and Klebsiella. This is due to the adhesion and utilization of the fucosylated glycocalyx on the intestinal mucosa by these pathogens, enabling some pathogens to utilize fucose.

[0047] Table 2 Prebiotic utilization value of each strain

[0048]

[0049] Example 4 Calculation of the probiotic activity of prebiotics based on multiple strains. The 8 strains were grouped as shown in Table 3.

[0050]

[0051] The beneficial activity of prebiotics was calculated according to the groups in Table 1. The formula is as follows:

[0052]

[0053] The mean of the beneficial bacteria growth value divided by the mean of the commensal bacteria and potential pathogenic bacteria growth value plus the mean of the beneficial bacteria prebiotic utilization value divided by the mean of the commensal bacteria and potential pathogenic bacteria prebiotic utilization value is the prebiotic activity value, as shown in Table 4, the prebiotic activity values ​​of different strains.

[0054]

[0055] Prebiotic activity calculations were performed using 4, 6, and 8 strains, respectively. Different strain groups produced different results, but as the number of included strains increased, the differences between groups decreased for the same number of strains. For the 4-strain calculation, the influence of different strain groupings on the calculation was significant, with large differences between groups. However, for the 6-strain calculation, while strain grouping still had a significant impact, the differences between groups were significantly reduced. The more strains included in the calculation, the more accurate the calculation. Therefore, Group E, which includes all 8 strains, provides the most accurate calculation of prebiotic activity of all groups.

[0056] Glucose significantly promoted the growth of all probiotic and potentially pathogenic strains. Using glucose as a benchmark, the calculated values ​​for galacto-oligosaccharides, fucoidan oligosaccharides, and 2'-fucosyllactose were higher than for glucose, while the calculated value for fucose was lower. Therefore, all prebiotics except fucose are considered high-quality prebiotics. Fuco-oligosaccharides had a significantly higher calculated value than the other prebiotics, demonstrating their ability to promote the growth of most probiotics while not supporting the growth of a wide range of potential pathogens, making them a broad-spectrum prebiotic. While 2'-fucosyllactose promoted the growth of only one probiotic, Bifidobacterium, it strictly did not support the growth of potential pathogens, resulting in the second highest calculated value. Galacto-oligosaccharides promoted the proliferation of most probiotics but also had growth-promoting effects on most pathogens, ranking third. Fucose is not suitable as a prebiotic because it does not proliferate probiotics but is utilized by potential pathogens, resulting in a lower calculated value than glucose.

[0057] Calculations on four prebiotics showed that the calculation of prebiotics based on multiple strains was more accurate, effective and objective in evaluating the prebiotic activity. The high or low calculated value of prebiotic activity could comprehensively reflect the prebiotic properties of a certain prebiotic, providing an objective detection method for the selection and development of prebiotics.

[0058] While the present invention has been disclosed above with reference to preferred embodiments, this is not intended to limit the present invention. Persons skilled in the art will readily appreciate that various modifications and variations can be made without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be determined by the claims.

Claims

1. A method for detecting beneficial activity based on multiple strains, characterized by: The steps include: Step 1: selecting strains to be tested for beneficial activity, the strains including beneficial bacteria and pathogenic bacteria, and the number of the strains is greater than 3; Step 2: The selected strains were cultured using the prebiotic to be evaluated as the sole carbon source in the culture medium, with glucose carbon source culture medium as the positive control group and sugar-free culture medium as the blank group; Step 3: Obtain the growth values ​​of all strains in step 2, draw a growth curve based on the OD600 absorbance value at each time point during the culture process, calculate the area under the growth curve, and then calculate the growth value of each strain; Step 4: Obtain the prebiotic utilization value of the strain, perform thin layer chromatography analysis on the supernatant of the bacterial liquid before and after cultivation of each strain, calculate the prebiotic consumption in the thin layer chromatography spectrum, and further calculate the prebiotic utilization value of each strain; The calculation method of the strain growth value in step 3 is as follows: the total number of strains is n, the strain number is i, 1≤i≤n, the prebiotic is named X, the blank group is named M, and the OD of the strain i is fermented for 0h, 12h, 24h, 36h, 48h until the fermentation end point th 600 The growth curve was drawn with the OD at 0 h. 600 The value is the baseline, and the area under the integrated 0-th growth curve is the value The growth value G is the area of ​​the prebiotic group (X) minus the area of ​​the blank group (M). i ; The calculation method of the prebiotic utilization value in step 4 is as follows: The total number of strains is n, the strain number is i, the prebiotic is named X, and thin layer chromatography is performed at fermentation 0h and fermentation end point th. The optical density value of th in the thin layer chromatography of the prebiotic is calculated using Image J software. and optical density value at 0h Subtract and divide by the optical density value at 0h Prebiotic utilization value U i ; Step 5, calculating the prebiotic activity using the growth value in step 3 and the prebiotic utilization value in step 4; The calculation method of the beneficial activity in step 5 is as follows: the total number of beneficial bacteria is n, each strain is numbered i, the total number of potential pathogens is N, each strain is numbered k, and the mean of the beneficial bacteria growth value is Divide by the mean of the potential pathogen growth value The average utilization value of beneficial bacteria and prebiotics Divided by the mean of the prebiotic utilization value of potential pathogens The prebiotic activity value P can be obtained; 。 2. The method for detecting beneficial activity of multiple strains according to claim 1, characterized in that: The beneficial bacteria include one or more of Bifidobacterium, Lactobacillus, Lactococcus, Streptococcus, Clostridium, and Enterococcus, and the pathogenic bacteria include one or more of Escherichia coli, Salmonella, Staphylococcus, Klebsiella, Vibrio, or Bacillus.

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